Method for specifically marking a protein

ABSTRACT

The present invention relates to a method for the specific labeling of a protein containing selenocyst(e)ine and/or cyst(e)ine groups, comprising the following steps: 
     at least one incubation of a protein-containing sample with at least one modifying agent specific for selenocyst(e)ine and/or cyst(e)ine groups, followed by 
     at least one further incubation of said protein-containing sample with at least one labeling agent specific for selenocyst(e)ine and/or cyst(e)ine groups; 
     wherein at least one substance interacting with said protein is added prior to and/or during and/or after at least one of said incubations.

The present invention relates to a method for the specific labeling of aprotein containing selenocyst(e)ine and/or cyst(e)ine groups.

The examination of receptors, such as the G protein coupled receptors,the polypeptide chain of which folds into a structure with sevenmembrane-spanning helices is of great importance in terms of bothscience and economy. These so-called 7-transmembrane receptors are alarge group of receptors which are very old phylogenetically and theamino acid sequence of which contains characteristic cysteines. Thecorresponding ligands belong to a wide variety of chemical classes ofsubstances and molecular sizes, from lipids, metal ions and monoaminesto peptides and proteins. However, to date, little is known aboutpossible structural changes of such receptors which represent the linkbetween ligand binding and signal transduction.

It is believed that ligand-induced conformational changes of the7-transmembrane receptors are involved in the initial step of thedissociation of the heterotrimeric G protein complex with exchange ofGDP/GTP. Schwartz and Rosenkilde (TiPS, Vol. 17, 1996) present a modelin which the capability of stabilizing an active receptor conformationis mentioned as the only requirement for a new agonist, resulting in theconclusion that a common "lock" for all the "keys" in the superfamily of7-transmembrane receptors does not exist.

Generally, in the examination of proteins, but also, in particular, inthe investigation of the signal cascade, cysteine-specific reagents areused in order to shed light upon the functional and structuralproperties of the cysteines (Creighton, "Proteins", Freeman & Co.,1984). Korner et al. (The Journal of Biological Chemistry, Vol. 257, No.7, pp. 3389-3396, 1982) describe a method for examining the β₂-adrenergic receptor using N-ethylmaleinimide (NEM). In order to reactthiol groups which are already exposed prior to the actual experiment,the membrane samples were pretreated with NEM in the presence of theantagonist propranolol in a first process step. In a further processstep, they were incubated with [³ H]isoproterenol with addition of NEM.In particular, the authors come to the conclusion that NEM interacts,not with the receptor itself, but with an associated component, the Gprotein. According to Korner et al., specific thiol groups of the Gprotein are being exposed when the G protein interacts with thehormone-activated receptor. Accordingly, the method of Korner et al. isnot suitable for labeling the actual receptor.

Andre et al. (Biochemical Pharmacology, Vol. 31, No. 22, 3657-3662,1982) describe the effect of NEM on agonist-induced conformationalchanges of the β-adrenergic receptor. The process performed on membranepreparations is characterized by three incubation steps: incubation withNEM, followed by incubation with NEM in the presence of the agonist(-)-isoproterenol, followed by incubation with the labeled antagonist(-)-[³ H]DHA. Andre et al. observed that the effects brought about byNEM can also be induced by GTP in their experiments, so that evidently Gproteins are critically involved.

Lipson et al. (Biochemistry 1986, 25, 5678-5685) describe a method forexamining the glucagon receptor N protein complex usingN-ethylmaleinimide and other reagents which alkylate thiols. It could bedemonstrated, for example, that the presence of NEM prior to, during orafter the association reaction of ¹²⁵ I-glucagon with partiallypurified, protein-containing liver membranes promotes the release ofbound hormone in a subsequent dissociation reaction.

In a survey article, in which the use of the alkylating agentN-ethylmaleinimide (NEM) in the investigation of G protein coupledreceptors was last mentioned (Lefkowitz et al., Ann. Rev. Biochem. 52,159-86, 1983), the authors note that it is uncertain whether thecritical sulfhydryl groups are localized on the receptor, on the Gprotein, or even on both components.

Today, the particular importance of the cysteines in the αβ-heterotrimerof the G protein is generally known. Corresponding examinations havebeen performed, e.g., by Garcia-Higuera (J. Biol. Chem., Vol. 257, No.1, 528-535, 1996) using crosslinking agents and by SDM (site-directedmutagenesis).

Li et al. (The Journal of Biological Chemistry, Vol. 267, No. 11,7570-7575, 1992) examined the importance of thiol groups to the bindingof the neuropeptide Y to the Y₂ receptor.

Examinations regarding the importance of disulfide bridges and thiolgroups to the binding of the thyrotropin-releasing hormone (TRH) to theTRH receptor have been performed by Cook et al. (Endocrinology, Vol.137, No. 7, 2851-2858, 1996). Dithiothreitol (DTT), a disulfide-bridgereducing agent, and p-CMB, a thiol-group blocking substance, reduce thespecific TRH binding in a dose-dependent way.

Approaches to the examination of conformational changes of G proteincoupled receptors are often indirect in nature, i.e., for example, theeffect of the receptor conformation on the GTPase activity of the Gprotein or the activity of effector enzymes is examined.

Gether et al. (The Journal of Biological Chemistry, Vol. 270, No. 47,issue of November 24, pp. 28268-28275, 1995), however, describe a methodfor the fluorescent labeling of the purified β₂ -adrenergic receptorwith the purpose of enabling ligand-induced conformational changes ofthis G protein coupled receptor to be directly recognized. Thus, thereceptor expressed in SF-9 insect cells was first subjected topurification, prior to being labeled with the cysteine-specificfluorescent markerN,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine(IANBD). The fluorescence of IANBD is highly dependent on the polarityof the dye environment and can therefore be employed as an indicator ofconformational changes of the receptor. Thus, a slight decrease influorescent emission could be observed upon the binding of the agonistisoproterenol to the β₂ -adrenergic receptor. A drawback of the methoddescribed by Gether et al. is, in particular, the extremely laboriousand time-consuming multistep purification of the receptor.

A great number of survey articles on the superfamily of nuclearreceptors have appeared just recently (Cell, Vol. 83, 835-839, 841-850,851-857, 1995), so that the function of these receptors shall not befurther dealt with in the present application.

It has been the object of the present invention to provide a method forthe specific labeling of protein-containing samples, such as receptors,especially transmembrane receptors, but also soluble receptors.

The object of the invention is achieved by a method according toclaim 1. Advantageous embodiments of the invention are described in thesubclaims.

The method according to the invention is characterized by the followingprocess steps:

at least one incubation of a protein-containing sample with at least onemodifying agent specific for selenocyst(e)ine and/or cyst(e)ine groups,followed by

at least one further incubation of said protein-containing sample withat least one labeling agent specific for selenocyst(e)ine and/orcyst(e)ine groups;

wherein at least one substance interacting with said protein is addedprior to and/or during and/or after at least one of said incubations.

The groups mentioned may be present in the protein essentially either inreduced form (selenocysteines or cysteines) or in oxidized form(selenocystines or cystines). The spellings "selenocyst(e)ine" and"cyst(e)ine" mean that the groups mentioned can be present essentiallyin one of the redox states mentioned.

The method according to the invention makes use of the fact thatapparently inaccessible selenocyst(e)ines and/or cyst(e)ines of theprotein can be labeled through the activation or conformational changeof the protein induced by the addition of a substance during incubationwith a labeling agent specific for selenocyst(e)ine and/or cyst(e)inegroups, or the degree of labeling of the protein and/or properties ofthe labeling agent are changed thereby.

For the detection of a thus induced activation or conformational change,it is desirable to modify the selenocyst(e)ines and/or cyst(e)inesalready accesible, in particular, prior to the addition of saidsubstance interacting with the protein in order to reduce the backgroundand to increase the detection efficiency. This is achieved by the one ormore additions of a modifying agent specific for selenocyst(e)ine and/orcyst(e)ine groups according to the invention. Thus, when the methodaccording to the invention is applied to 7-transmembrane receptors,negative allosteric effects of the G protein on the receptor whichaccelerate the agonist dissociation can be advantageously suppressed, orinterfering cysteine proteases which can cause degradation of theagonist receptor complex can be inactivated. Said modifying and labelingagents specific for selenocyst(e)ine and/or cyst(e)ine groups may beadded in stoichiometric amounts. According to another preferredembodiment of the method according to the invention, they are added inexcess. Excess modifying agent can be advantageously removed prior tothe incubation of the protein-containing sample with a labeling agent,especially by centrifugation or by washing steps, or it may beinactivated by the addition of chemicals containing cysteine,selenocysteine or SH groups.

As an advantage over the prior art, the method according to theinvention is characterized by being particularly suitable for use inhigh-throughput screening due to the use of a unique labeling agent andenabling the detection of new ligands for protein-containing samples,especially known ones, or vice versa. An accelerated functionalvalidation of orphan receptors, especially 7-transmembrane orphanreceptors, as target/lead systems and of known receptors in pharmascreening for new agonists or antagonists by a general test procedure isof utmost economic importance. Thus, for example, agonists for an orphanreceptor with unknown function can be identified from a ligand libraryusing the method according to the invention. The method according to theinvention allows to differentiate between agonists and antagonists.Thus, a detection of antagonists can be achieved due to the inhibitionof the agonist-induced labeling or by the detection of asubstrate/inhibitor interaction.

The method according to the invention can be used in a screeningprocedure for determining substances interacting with a protein to beexamined. Such substances may be derived, for example, from extracts ofnatural substances. Currently, commercial vegetable drugs mostly containraw extracts, chromatographic fractions, mixtures or emulsions ofsubstances. It is generally considered that at least one quarter of thedrugs employed today in industrial countries are of vegetable origin orare prepared after a model of substances contained in plants. However,the substances may also be derived from the reservoir of substancesprovided by combinatory chemistry. Using this method, several millionsof structurally related substances can be produced in a very short time.These are often preselected compounds for which it is to be expected,from the properties of the starting materials, that at least some ofthem exhibit the desired activity in a more or less pronounced form. Themost effective compounds can be selected by screening the resulting poolof compounds using the method according to the invention.

Using the method according to the invention, it is also possible to testproteins, especially those which have not been further characterized,for a potential interaction with substances, especially known ones, in ascreening procedure.

Medicinal substances preferably act on enzymes, receptors, transporters,ion channels and signal proteins. Some inhibit the reactions catalyzedby enzymes, while others bind to receptors and thereby either, asagonists, cause the same effect as the endogenous messengers, or, asantagonists, have the very opposite effect by preventing normal ligandsfrom accessing the site or impeding the formation of a giventhree-dimensional structure of the receptor. Similarly, withtransporters, the substances actually to be transported are displaced.In the case of ion channels, the drug stabilizes either the open or theclosed form. Finally, signal proteins control the activitty of enzymes,receptors or ion channels and can also be influenced by medicinalsubstances. In all these cases, it is critical that the medicinalsubstance communicate with the biological structures or block theinteraction with the actually intended agent.

The method according to the invention offers the possibility to testsubstances as well as proteins for whether they interact with eachother.

It may be desirable to adjust the incubation times to the kinds ofmodifying and labeling agents specific for selenocyst(e)ine and/orcyst(e)ine groups. Further, it is possible, after incubation of theprotein-containing sample with the modifying agents specific forselenocyst(e)ine and/or cyst(e)ine groups, to add only the substancefirst for preequilibration and to add the labeling agent specific forselenocyst(e)ine and/or cyst(e)ine groups only at a later time. Theaddition of the labeling agent may be done once or several times.However, it may also be desirable first to add the substance interactingwith the protein to the sample and to add the modifying agent and thelabeling agent in subsequent steps. Similarly, the other times ofaddition of the substance interacting with the protein as mentioned inclaim 1 may also be advantageous.

According to another embodiment of the method according to theinvention, transmembrane receptors, in particular, such as G proteincoupled 7-transmembrane receptors, are specifically labeled, or aligand-induced activation and/or deactivation and/or conformationalchange of these receptors is detected.

Reagents specific for cyst(e)ine groups or selenocyst(e)ine groups areknown from the literature (Creighton, "Proteins", Freeman & Co., 1984)and can accordingly be employed in the method according to theinvention. In this connection, experiments have been performed withvarious reagents specific for selenocyst(e)ines or cyst(e)ines(including iodoacetate, iodoacetamide, diamide, monovalent and divalentmaleinimides, such as NEM, various metal compounds, DTT, NaBH₄,pyridoxal phosphate). Preferably, alkylating reagents, such asN-ethylmaleinimide (NEM), may be used in the method according to theinvention. It may further be preferred to perform an incubation of theprotein-containing sample with a reducing agent, such asβ-mercaptoethanol or dithiothreitol, in order to influence cysteineswhich are linked to one another through disulfide bridges.

Through the combination of alkylating and reducing cyst(e)ine-reactivereagents, it is possible to further optimize the reaction conditions.Reagents of different hydrophobicities and sizes have different membranepermeabilities, which may be considered in the method according to theinvention.

The substitution of cysteines by selenocysteines in proteins isdescribed in the more recent literature (Muller et al., Biochemistry,33, 3404-3412, 1994; Ursini et al., Methods in Enzymology, Vol. 252,38-53, Academic Press, 1995; Bjornstedt et al., Methods in Enzymology,Vol. 252, 209-219, Academic Press, 1995).

A "labeling agent specific for selenocyst(e)ine or cyst(e)ine groups"within the meaning of the invention includes any reagents which willreact with selenocyst(e)ine or cyst(e)ine and which contain a detectableportion. The detectable portion may be selected depending on thedetection system, e.g., haptens for immunological detection,multivalent, preferably divalent, functional groups for a cross-linkingreaction. Further, radio-labeled substances may also be used, ordetection may be effected using biotin/streptavidin or avidin. It may bepreferred to use biotin, as in Example 1, so that the specific labelingof a receptor can be detected by a Western blot using appropriateantibodies. The labeling agent specific for selenocyst(e)ine orcyst(e)ine may preferably be luminescent. Then, for the detection of thelabeling having been effected and/or an activation and/or deactivationand/or conformational change of the receptor, the method of fluorescencecorrelation spectroscopy (WO 94/16313) and other confocal fluorescencetechniques, as described in the publication WO 96/13744 and in theEuropean Patent Application 96 116 373.0, can be employed. The latterapplication suggests a method for analyzing samples by repeatedlymeasuring the number of photons per defined time interval in light whichis emitted, scattered and/or reflected by the particles in the sampleand determining the distribution of the number of photons in therespective time intervals, characterized in that the distribution of themolecular brightness of the particles is determined from thedistribution of the numbers of photons. There may also be used a methodfor analyzing samples which relies on the repeated measurement of thelength of time intervals between photons, determining the distributionof properties of the particles, such as the distribution of themolecular brightness, from the distribution of the lengths of the timeintervals. It may also be desirable to employ a detection method whichdetermines at least two properties of the labeled particles from atleast two-dimensional intermediary statistical data. Such a method isexplained in more detail in the European Patent Application 97 109353.9. It may further be preferred to employ a signal analyzing methodfor the detection of the labeling reaction, which method is described inthe German Patent Application 196 49 048.0 and in the InternationalPatent Application PCT/EP 97/06622. Said method is a method fordifferentiating or detecting particles in a sample in which severalclasses of particles may be present by identifying signal segments oftime-resolved, optical raw signals from the sample on the basis ofsingle photon detection (single pulse detection), wherein

the sample contains at least two classes of particles;

the sample is illuminated by a light source;

the optical raw signals emitted by the sample, which are derived from atleast one measuring volume element V, V≦10⁻¹² l, are detected with atleast one detector unit;

at least one particle generates a signal fraction during its residencein the measuring volume element;

a signal segment of the optical raw signals is determined by theparticle's actively and/or passively entering and then leaving again themeasuring volume element;

the optical raw signals are segmented into arbitrary segments;

at least one set of statistical data based on the optical raw signals isestablished for at least one arbitrarily chosen segment; and

said at least one set of statistical data or at least one combination ofseveral sets of statistical data is evaluated for the presence offeatures characteristic of the signal fraction from at least one classof particles.

Further, it may be preferred to detect the labeling reaction using themethod for determining predefined properties of target particles of asample medium as described in the German Patent Application 197 02914.0. The disclosures of the above mentioned patent applications,especially with respect to the detection and signal analyzing methodsand devices suitable therefor as explained therein, are incorporatedherein by reference.

The provision of the protein-containing sample can be done, inparticular, using (recombinant) cells, tissues, vesicles or artificialmembranes. Advantageously, the method according to the invention doesnot forcibly require purification of the components involved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of NEM on the interaction of thyrotropin (TSH)with the TSH receptor.

FIG. 2 shows the effect of DTT on the interaction of thyrotropin (TSH)with the TSH receptor.

FIG. 3 shows TSH binding curves.

FIG. 4 shows the results of Western Blots with respect to the detectionof the TSH FLAG receptor and the binding of biotin to the receptor.

FIG. 5 shows the results of the Western Blots after different NEM andTSH treatments of the TSH receptor.

FIG. 6 shows the effect of NEM on the interaction of a ¹²⁵ I-labeledantagonist (iodocyanopindolol, ICYP) with the β₂ -adrenergic receptor inthe presence of the agonist (-)-isoproterenol (100 nM).

FIG. 7 shows the effect of DTT on the interaction of a ¹²⁵ I-labeledantagonist (iodocyanopindolol, ICYP) with the β₂ -adrenergic receptor inthe presence of the agonist (-)-isoproterenol (100 nM).

FIG. 8 shows the effects of NEM and DTT on the radioligand receptorassay using the β₂ -adrenergic receptor.

FIG. 9 shows the results of Western Blots with respect to the detectionof the β₂ -adrenergic receptor and the binding of biotin to thereceptor.

FIG. 10 shows the effect of NEM on the interaction of ¹²⁵ I-labeledendothelin-1 with the endothelin (ETA) receptor.

FIG. 11 shows the effect of DTT on the interaction of ¹²⁵ I-labeledendothelin-1 with the endothelin (ETA) receptor.

FIG. 12 shows the effects of NEM and DTT on the radioligand ETA receptorassay using the ETA receptor.

FIG. 13 shows the results of Western Blots with respect to the detectionof the ETA receptor and the binding of biotin to the receptor.

FIG. 14 shows the effect of estradiol on the binding of NEM-biotin tothe human estrogen receptor α (hERα).

FIG. 15 shows the application of the method according to the inventionto the examination of the human estrogen receptor α (hERα).

FIG. 1 illustrates the effect of NEM on the interaction of TSH with theTSH receptor. ¹²⁵ I-labeled bovine TSH (B) or HeLa cells transfectedwith human TSH receptor (C) were incubated at room temperature with 3 mMNEM for 20 min. After dilution or washing steps, untreated receptor orradioligand was added. The assay was incubated at 30° C. over night, andthe receptor fraction was subsequently precipitated for determining thebound ¹²⁵ I-TSH. (D) shows the result of an assay performed in thepresence of 3 mM NEM, neither the hormone nor the receptor fractionhaving previously been pretreated with NEM. (A) shows the controlincubation without any NEM incubation.

FIG. 2 illustrates the effect of DTT on the interaction of TSH with theTSH receptor. ¹²⁵ I-labeled bovine TSH (B) or HeLa cells transfectedwith human TSH receptor (C) were incubated at room temperature with 1 mMDTT for 20 min. After dilution or washing steps, untreated receptor orradioligand was added. The assay was incubated at 30° C. over night, andthe receptor fraction was subsequently precipitated for determining thebound ¹²⁵ I-TSH. (D) shows the result of an assay performed in thepresence of 1 mM DTT, neither the hormone nor the receptor fractionhaving previously been pretreated with DTT. (A) shows the controlincubation without any DTT incubation.

FIG. 3 represents TSH binding curves with untreated receptor as acontrol (a), with a receptor pretreated with 3 mM NEM (b), with areceptor treated with 3 mM NEM only in the subsequent binding assay (c),and with a receptor which has been both pretreated with 3 mM NEM andtreated with 3 mM NEM in the assay (d).

FIG. 4 shows the results of the Western Blots with respect to thedetection of the TSH FLAG receptor as a control (anti-FLAG blot) as wellas the detection of the cysteine-specific labeling agent NEM-biotinbound to the TSH receptor. HeLa cells transfected with the TSH FLAGreceptor were incubated once with each of different NEM concentrationsfor 30 minutes (column 1: HeLa cells without TSH FLAG receptor, no NEMpretreatment; column 2: HeLa cells with TSH FLAG receptor, no NEMpretreatment; column 3: HeLa cells with TSH FLAG receptor, pretreatmentwith 100 μM NEM; column 4: HeLa cells with TSH FLAG receptor,pretreatment with 1 mM NEM; column 5: HeLa cells with TSH FLAG receptor,pretreatment with 10 mM NEM). After removing the NEM solutions andwashing the cells, the cells were incubated with 100 nM TSH and 1 mMNEM-biotin for one hour. After treatment with cell lysis buffer, the TSHFLAG receptor was immunoprecipitated by means of anti-FLAG antibodies.The immunoprecipitate was divided into two fractions which were bothfurther treated by SDS PAGE. The separated proteins were transferred tonitrocellulose membranes for the Western blots (anti-FLAG andanti-biotin). Detection was effected by using a second enzyme-coupledantibody (anti-Ig). The TSH FLAG receptor shows a characteristic patternof bands (anti-FLAG blot) consisting of a major band and two minorbands, which pattern remained unaffected by the NEM treatment. Thelabeling of the TSH receptor with NEM-biotin increases with increasingconcentrations of NEM in the pretreatment (anti-biotin blot). Higher NEMconcentrations can influence accordingly more cysteine SH groups alreadypresent, so that less NEM-biotin is lost due to non-specific bindingreactions on the cells. Consequently, relatively more NEM-biotin isavailable for the labeling of the SH groups made accessible by aligand-induced activation and/or conformational change of the receptor.

FIG. 5 shows the results of the Western Blots after different NEM andTSH treatments of the TSH receptor. The cells were divided into threefractions and pelletized with buffer (columns 1 to 3) or with 3 mM NEM(columns 4 to 6: incubation for 20 minutes; columns 7 to 9: incubationfor 20 hours) prior to incubation. Then, each sample was further treatedas follows: incubation for one hour with buffer (columns 1, 4 and 7),incubation for one hour with 100 nM TSH (columns 2, 5 and 8), orincubation for 22 hours with 100 nM TSH (columns 3, 6 and 9). This wasrespectively followed by incubation for one hour with 1 mM NEM-biotin.The samples were centrifuged, washed several times, lysed and, after SDSPAGE, examined by a Western blot. The TSH receptor wasimmunoprecipitated with anti-FLAG antibodies and examined for biotinbinding in the blot. Columns 1 and 2 show a weak NEM-biotin labeling ofthe receptor which can be increased by incubation with TSH for one hour(column 2). Columns 4 to 6 show, on the one hand, the increased labelingof the receptor brought about by incubation with NEM for 20 minutes and,on the other hand, furnish evidence of the higher labeling rate of thereceptor achieved by TSH incubation. The specificity of the TSH-inducedNEM-biotin labeling of the receptor is evidently lost during long NEMincubation times (columns 7 to 9).

FIG. 6 illustrates the effect of NEM on the interaction of a ligand withthe β₂ -adrenergic receptor. The agonist (-)-isoproterenol (B) and A431cells expressing the human β₂ -adrenergic receptor (C) were incubated atroom temperature with 3 mM NEM for 20 min. After dilution or washingsteps, untreated receptor or ligand was added in the presence of ¹²⁵I-ICYP. The assay was incubated at 30° C. for 40 min, and the receptorfraction was subsequently precipitated for determining the bound ¹²⁵I-ICYP. The pretreatment of the agonist or the receptor with NEM hasonly a small effect on the binding. (D) shows the result of an assayperformed in the presence of 3 nM NEM, neither the hormone nor thereceptor fraction having previously been pretreated with NEM. (A) showsthe control incubation without any NEM incubation.

FIG. 7 illustrates the but small effect of DTT on the interaction of aligand with the β₂ -adrenergic receptor. The agonist (-)-isoproterenol((B) and A431 cells expressing the human β₂ -adrenergic receptor (C)were incubated at room temperature with 8 mM DTT for 20 min. Afterdilution or washing steps, untreated receptor or ligand was added in thepresence of ¹²⁵ I-ICYP. The assay was incubated at 30° C. for 40 min,and the receptor fraction was subsequently precipitated for determiningthe bound ¹²⁵ I-ICYP. The pretreatment of the agonist or the receptorwith NEM has only a small effect on the binding. (D) shows the result ofan assay performed in the presence of 8 mM DTT, neither the hormone northe receptor fraction having previously been pretreated with DTT. (A)shows the control incubation without any DTT incubation.

¹²⁵ I-ICYP binding curves with the receptor in the presence of differentquantities of NEM (a) or DTT (b) are represented in FIG. 8.

FIG. 9 shows the results of the Western Blots with respect to thedetection of the β₂ -adrenergic receptor as a control (anti-β₂-adrenergic receptor blot) as well as the detection of thecysteine-specific labeling agent NEM-biotin bound to the β₂ -adrenergicreceptor. A431 cells were incubated for 10 minutes with (columns 4 and5) and without (columns 1, 2 and 3) NEM (10 mM). After removing the NEMsolutions and washing the cells, the cells were incubated with 100 μM(-)-isoproterenol for 5 min (columns 3 and 5). This was followed by onehour of incubation with 1 mM NEM-biotin (columns 2 to 5), wherein(-)-isoproterenol was still present for columns 3 and 5. After treatmentwith RIPA buffer, the β₂ -adrenergic receptor was immunoprecipitated bymeans of anti-β₂ -adrenergic receptor antibodies. The immunoprecipitatewas divided into two fractions which were both further treated by SDSPAGE. The separated proteins were transferred to PVDF membranes for theWestern blots (anti-β₂ -adrenergic receptor blot and anti-biotin blot).Detection was effected by using a second enzyme-coupled antibody(anti-Ig). The β₂ -adrenergic receptor shows a characteristic pattern ofbands (anti-β₂ -adrenergic receptor blot) consisting of a specific band,which pattern remained unaffected by the NEM treatment. The labeling ofthe receptor with NEM-biotin can only be detected if the agonist hasbeen added. SH groups made accessible by a ligand-induced activationand/or conformational change of the receptor are now evidently labeledby NEM-biotin. A comparison between the columns 3 and 5 shows that theNEM pretreatment advantageously increases the specificity of thedetection reaction.

FIG. 10 illustrates the effect of NEM on the interaction of a ligandwith the endothelin receptor. ¹²⁵ I labeled endothelin-1 (B) and humanendothelin receptor (C) were incubated at room temperature with 4 mM NEMfor 20 min. After dilution or washing steps, untreated receptor orradioligand was added. The assay was incubated at 30° C. over night, andthe receptor fraction was subsequently precipitated for determining thebound ¹²⁵ I-endothelin. The pretreatment of the agonist or the receptorwith NEM has only a small effect on the binding. (D) shows the result ofan assay performed in the presence of 4 mM NEM, neither the hormone northe receptor fraction having previously been pretreated with NEM. (A)shows the control incubation without any NEM incubation.

FIG. 11 illustrates the effect of DTT on the interaction of a ligandwith the endothelin receptor. ¹²⁵ I labeled endothelin-1 (B) and humanendothelin receptor (C) were incubated at room temperature with 0.5 mMDTT for 20 min. After dilution or washing steps, untreated receptor orradioligand was added. The assay was incubated at 30° C. over night, andthe receptor fraction was subsequently precipitated for determining thebound ¹²⁵ I-endothelin. The pretreatment of the agonist or the receptorwith DTT has an effect on the binding. (D) shows the result of an assayperformed in the presence of 0.5 mM DTT, neither the hormone nor thereceptor fraction having previously been pretreated with DTT. (A) showsthe control incubation without any DTT incubation.

¹²⁵ I-endothelin binding curves with the receptor in the presence ofdifferent quantities of NEM (a) or DTT (b) are represented in FIG. 12.The disulfide bridges of ¹²⁵ I-endothelin are evidently disrupted athigh DTT concentrations.

FIG. 13 shows the results of the Western Blots with respect to thedetection of the endothelin receptor as a control (anti-endothelinreceptor blot) as well as the detection of the cysteine-specificlabeling agent NEM-biotin bound to the endothelin receptor. RAT2 cellswere incubated for 10 minutes with (columns 4 and 5) and without(columns 1, 2 and 3) NEM (10 mM).

After removing the NEM solutions and washing the cells, the cells wereincubated with 3 nM endothelin for 10 min (columns 3 and 5). This wasfollowed by two hours of incubation with 1 mM NEM-biotin (columns 2 to5), wherein endothelin was still present for columns 3 and 5. Aftertreatment with RIPA buffer, the endothelin receptor wasimmunoprecipitated by means of anti-endothelin receptor antibodies. Theimmunoprecipitate was divided into two fractions which were both furthertreated by SDS PAGE. The separated proteins were transferred to PVDFmembranes for the Western blots (anti-endothelin receptor andanti-biotin). Detection was effected by using a second enzyme-coupledantibody (anti-Ig). The endothelin receptor shows a characteristicpattern of bands (anti-endothelin receptor blot) consisting of aspecific band, which pattern remained unaffected by the NEM treatment.The labeling of the endothelin receptor with NEM-biotin can only bedetected if the agonist has been added. SH groups made accessible by aligand-induced activation and/or conformational change of the receptorare now evidently labeled by NEM-biotin. In this case too, it is foundthat the NEM pretreatment advantageously increases the specificity ofthe detection reaction (columns 3 and 5).

FIG. 14 shows the effect of estradiol on the binding of NEM-biotin tothe human estrogen receptor. Further explanations are given in Example4.

FIG. 15 shows the effect of estradiol on the accessibility ofcyst(e)ines in the estrogen receptor hERα. The method according to theinvention was performed here in the following order: addition of ligand(estradiol), addition of modifying agent (NEM), addition of labelingagent (NEM-biotin). Further explanations are given in Example 4.

EXAMPLE 1

Specific labeling and/or determination of conformational changes of theTSH receptor using biotinylated NEM as a cysteine-reactive labelingagent (cf. FIGS. 1 to 5).

Cells containing the TSH receptor provided with a FLAG epitope wereincubated at room temperature in assay buffer (10 mM potassium phosphatewith 0.1 g/l BSA, 0.01 g/l sodium azide, and 0.01 g/l phenol red, pH7.4) with 3 mM NEM for 30 min. The excess reagent was then removed bycentrifugation (1000× g, 5 min). This was followed by incubation overnight with 100 μM TSH in the presence of 300 μM biotinylated NEM. Thereaction efficiency of biotinylated NEM was previously established in aseparate assay and corresponded to that of NEM. Then, the receptorfraction was centrifuged twice (2×10000× g, 2 min), the supernatantswere removed, and the cells were covered with 1 ml of cold protein lysisbuffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1 g/l Nonident P-40 with1 tablet of Boehringer "Complete Protease Inhibitor Complex"). After 20minutes of incubation at 4° C. on ice, the cells were scraped off, andundesired components were pelletized by centrifugation (10000× g, 2min). To 250 μl of the cell lysate was added 5 μl of the anti-FLAGantibody, followed by incubation at 4° C. for 60 min. Aftercentrifugation (10000× g, 15 min, 4° C.) and removing the supernatant,protein A sepharose beads were added. This was followed by another 60minutes of incubation at 4° C., followed by centrifugation (6000× g, 30s). Then, several washing steps (20 mM Tris-HCl, pH 8, 100 mM NaCl, 0.05g/l Nonident P-40) were performed with repeated centrifugations. Thepellet was resuspended in 150 μl of washing buffer. Then, to 40 μl ofthis sample was added 8 μl of sample buffer (50 mM Tris-HCl, pH 6.8, 4%SDS, 12% glycine, 2% β-mercaptoethanol, 0.01% SERVA Blue G). The sampleswere then boiled for 3 to 5 min, briefly centrifuged and subsequentlyseparated by discontinuous 10% SDS PAGE. After the electrophoresis, thegel was removed, washed in blotting buffer (48 mM Tris, 39 mM glycine,20% methanol, 0.037% SDS) and blotted onto a PVDF membrane. The membranewas dried over night at room temperature, and then 20% methanol wasadded. For reducing the background, the membrane was blocked for 1 hour(1% w/v casein hydrolysate). The blocking buffer was subsequentlyremoved. The membrane was incubated with 10 ml of a suitable firstantibody dilution (anti-biotin 1:500, and for control:anti-FLAG 1:1000,each in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20, 0.1% caseinhydrolysate) for 1 hour, washed three times for 10 min each (20 mMTris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) and then incubated withthe respective second antibody (1:4000 as peroxidase conjugates, in 20mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20, 0.1% caseinhydrolysate) for 1 hour. After several washings (20 mM Tris-HCl, pH 7.6,137 mM NaCl, 0.05% Tween 20), the last washing being performed withwater, 5 ml each of solutions 1 and 2 was added, and the blot was soakedtherein for 60 seconds. Then, a film was placed thereon. The expositiontime was determined by the signal intensity.

EXAMPLE 2

Specific labeling and/or determination of conformational changes of theβ₂ -adrenergic receptor using biotinylated NEM as a cysteine-reactivelabeling agent (cf. FIGS. 6 to 9).

A431 fibroblasts expressing the β₂ -adrenergic receptor were incubatedat room temperature in DMEM (Dulbecco's modified Eagle's medium) with 1%BSA and 10 mM NEM for 10 min. The excess reagent was then removed bywashing with DMEM with 1% BSA. This was followed by incubation for onehour with 100 μM (-)-isoproteronol in the presence of 1 mM biotinylatedNEM. The reaction efficiency of biotinylated NEM was previouslyestablished in a separate assay. Then, the cells were removed from theculture flask by slightly scraping, and then the receptor fraction wascentrifuged twice (2×10000× g, 2 min), the supernatants were removed,and the cells were covered with 1 ml of cold RIPA buffer (0.1% SDS, 1%Triton X100, 1% sodium deoxycholate, 0.15 M NaCl, 0.01 M Tris-HCl, pH7.4, 1 mM EDTA, 1 tablet of Boehringer "Complete Protease InhibitorComplex"). After 20 minutes of incubation at 4° C. on ice, the cellswere scraped off, and undesired components were pelletized bycentrifugation (10000× g, 2 min). To 1000 μl of the cell lysate wasadded 10 μl of Normal Rabbit Serum, followed by incubation at 4° C. for60 min. After centrifugation (10 000× g, 15 min, 4° C.) and removing thesupernatant, protein A sepharose beads were added. This was followed byanother 60 minutes of incubation at 4° C. After centrifugation (10 000×g, 15 min, 4° C.) and removing the supernatant, 4 μg of the specificanti-β₂ -adrenergic receptor antibody was added. This was followed byanother 60 minutes of incubation at 4° C. After centrifugation (10000×g, 15 min, 4° C.) and removing the supernatant, protein A sepharose wasadded. This was followed by centrifugation (6000× g, 30 s). Then,several washing steps (0.1% SDS, 1% Triton X100, 1% sodium deoxycholate,0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4, 1 mM EDTA, 1 tablet of Boehringer"Complete Protease Inhibitor Complex" ) were performed with repeatedcentrifugations. The pellet was resuspended in 30 μl of sample buffer(50 mM Tris-HCl, pH 6.8, 4% SDS, 12% glycine, 2% β-mercaptoethanol,0.01% SERVA Blue G). The samples were then boiled for 3 to 5 min,briefly centrifuged and subsequently separated by discontinuous 10% SDSPAGE. After the electrophoresis, the gel was removed, washed in blottingbuffer (48 mM Tris, 39 mM glycine, 20% methanol, 0.037% SDS) and blottedonto a PVDF membrane. The membrane was dried over night at roomtemperature, and then 20% methanol was added. For reducing thebackground, the membrane was blocked for 1 hour (1% w/v caseinhydrolysate). The blocking buffer was subsequently removed. The membranewas incubated with 10 ml of a suitable first antibody dilution(anti-biotin 1:2500, and for control: anti-β₂ -adrenergic receptor1:1000, each in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20,0.1% casein hydrolysate) for 1 hour, washed three times for 10 min each(20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) and then incubatedwith the respective second antibody (1:2500 as peroxidase conjugates, in20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20, 0.1% caseinhydrolysate) for 1 hour. After several washings (20 mM Tris-HCl, pH 7.6,137 mM NaCl, 0.05% Tween 20), the last washing being performed withwater, 5 ml each of ECL solutions 1 and 2 was added, and the blot wassoaked therein for 60 seconds. Then, a film was placed thereon. Theexposition time was determined by the signal intensity.

EXAMPLE 3

Specific labeling and/or determination of conformational changes of theendothelin receptor using biotinylated NEM as a cysteine-reactivelabeling agent (cf. FIGS. 10 to 13).

RAT2 fibroblasts expressing the endothelin receptor were incubated atroom temperature in DMEM (Dulbecco's modified Eagle's medium) with 1%BSA and 10 mM NEM for 10 min. The excess reagent was then removed bywashing with DMEM with 1% BSA. This was followed by incubation for 2hours with 3 nM endothelin in the presence of 1 mM biotinylated NEM. Thereaction efficiency of biotinylated NEM was previously established in aseparate assay. Then, the cells were removed from the culture flask byslightly scraping, and then the receptor fraction was centrifuged twice(2×10000× g, 2 min), the supernatants were removed, and the cells werecovered with 1 ml of cold RIPA buffer (0.1% SDS, 1% Triton X100, 1%sodium deoxycholate, 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4, 1 mM EDTA, 1tablet of Boehringer "Complete Protease Inhibitor Complex"). After 20minutes of incubation at 4° C. on ice, the cells were scraped off, andundesired components were pelletized by centrifugation (10000× g, 2min). To 1000 μl of the cell lysate was added 10 μl of either NormalRabbit Serum or Normal Sheep Serum (depending on the primaryanti-receptor antibody employed), followed by incubation at 4° C. for 60min. After centrifugation (10 000× g, 15 min, 4° C.) and removing thesupernatant, protein G sepharose beads were added. This was followed byanother 60 minutes of incubation at 4° C. After centrifugation (10 000×g, 15 min, 4° C.) and removing the supernatant, 4 μg of the specificanti-endothelin receptor antibody was added. This was followed byanother 60 minutes of incubation at 4° C. After centrifugation (10 000×g, 15 min, 4° C.) and removing the supernatant, protein G sepharose wasadded. This was followed by centrifugation (6000× g, 30 s). Then,several washing steps (0.1% SDS, 1% Triton X100, 1% sodium deoxycholate,0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4, 1 mM EDTA, 1 tablet of Boehringer"Complete Protease Inhibitor Complex") were performed with repeatedcentrifugations. The pellet was resuspended in 30 μl of sample buffer(50 mM Tris-HCl, pH 6.8, 4% SDS, 12% glycine, 2% β-mercaptoethanol,0.01% SERVA Blue G). The samples were then boiled for 3 to 5 min,briefly centrifuged and subsequently separated by discontinuous 10% SDSPAGE. After the electrophoresis, the gel was removed, washed in blottingbuffer (48 mM Tris, 39 mM glycine, 20% methanol, 0.037% SDS) and blottedonto a PVDF membrane. The membrane was dried over night at roomtemperature, and then 20% methanol was added. For reducing thebackground, the membrane was blocked for 1 hour (1% w/v caseinhydrolysate). The blocking buffer was subsequently removed. The membranewas incubated with 10 ml of a suitable first antibody dilution(anti-biotin 1:2500, and for control: anti-endothelin receptor 1:1000,each in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20, 0.1% caseinhydrolysate) for 1 hour, washed three times for 10 min each (20 mMTris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) and then incubated withthe respective second antibody (1:2500 as peroxidase conjugates, in 20mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20, 0.1% caseinhydrolysate) for 1 hour. After several washings (20 mM Tris-HCl, pH 7.6,137 mM NaCl, 0.05% Tween 20), the last washing being performed withwater, 5 ml each of ECL solutions 1 and 2 was added, and the blot wassoaked therein for 60 seconds. Then, a film was placed thereon. Theexposition time was determined by the signal intensity.

EXAMPLE 4

Specific labeling and/or determination of conformational changes of thehuman estrogen receptor (hERα) using biotinylated NEM as acysteine-reactive labeling agent.

The human estrogen receptor α (hERα) was expressed as a recombinantprotein in baculovirus infected Sf9 cells and purified by 17β-estradiolagarose chromatography. 2 μg of the affinity-purified hERα in 50 mMTris, pH 7.5, 10% glycerol, 0.5 M KCl, 1 mM EDTA, 2 mM DTT, 1 mM sodiumvanadate and 0.02% sodium azide was diluted to a concentration of 2ng/μl in 1 ml of TEG buffer (10 mM Tris, 10% glycerol, 1 mM EDTA, 0.5 mMDTT) at pH 8.0 and divided into two equal fractions. To one of thefractions was added 50 ml of TEG buffer ("minus estradiol", -E₂), and tothe other fraction was added 50 μl of 17β-estradiol in TEG buffer ("plusestradiol", +E₂) to reach an estradiol concentration of 200 μM.Saturation of the ligand binding domain was achieved by 20 minutes ofincubation at room temperature. Then, the incubation was cooled down.All subsequent steps were performed at 4° C. in order to reduce thedissociation rate of the hormone receptor complex to a low level. The"minus" and "plus" estradiol aliquots were further divided into 2fractions each. To one of the "minus"/"plus" pairs was added 10 μl of a55 mM NEM solution (in ethanol) to reach an effective concentration of2.2 mM. To the other "minus"/"plus" pair was added 10 μl of a 55 mMNEM-biotin solution (in DMSO) to reach an effective concentration of 2.2mM; the time dependence of the alkylation reaction was to be followed.This moment is considered time "0". 50 μl of the different incubationswere removed at particular times (up to 45 min), and 10 μl of samplebuffer (50 mM Tris-HCl, pH 6.8, 4% SDS, 12% glycine, 2%β-mercaptoethanol, 0.01% SERVA Blue G) was added. The molar excess ofβ-mercaptoethanol prevented further alkylation of the receptor andinactivated residual NEM and NEM-biotin in the reaction mixture. Thereaction rate for the modification of the available cysteine sulfhydrylgroups was followed by their biotinylation and detected by means ofsubsequent Western blots (FIG. 14). The "minus"/"plus" pair to which NEMhad been added was also subjected to PAGE and blotted onto PVDFmembrane. The membrane was dried over night at room temperature, andthen 20% methanol was added. hERα was biotinylated directly on the PVDFmembrane (1.7 mM NEM-biotin in TEG buffer, 20 min); see FIG. 15. Thereaction was quenched by repeatedly washing the membrane with TBS-T (20mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20). For reducing thebackground, the membrane was blocked for 1 hour (1% w/v caseinhydrolysate). The blocking buffer was subsequently removed. The membranewas incubated with 10 ml of a suitable antibody dilution (anti-biotin1:2500 as a peroxidase conjugate in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl,0.05% Tween 20, 0.1% casein hydrolysate) for 1 hour, washed three timesfor 10 min each (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20)and finally washed with water. Then, 5 ml each of ECL solutions 1 and 2was added, and the blot was soaked therein for 60 seconds. Then, a filmwas placed thereon. The exposition time was determined by the signalintensity.

What is claimed is:
 1. A method for the specific labeling of a proteincontaining selenocyst(e)ine and/or cyst(e)ine groups, comprising thefollowing steps:at least one incubation of a protein-containing samplewith at least one modifying agent specific for selenocyst(e)ine and/orcyst(e)ine groups, followed by at least one further incubation of saidprotein-containing sample with at least one labeling agent specific forselenocyst(e)ine and/or cyst(e)ine groups; wherein at least onesubstance interacting with said protein is added prior to and/or duringand/or after at least one of said incubations.
 2. The method accordingto claim 1, characterized in that said modifying agent specific forselenocyst(e)ine and/or cyst(e)ine groups will alkylate these groups. 3.The method according to any of claim 1, characterized in that saidlabeling agent specific for selenocyst(e)ine and/or cyst(e)ine groupswill alkylate these groups.
 4. The method according to claim 1,characterized in that said modifying agent is N-ethylmaleinimide,dithiothreitol, dithioerythritol, β-mercaptoethanol, iodoacetamide,iodoacetate, diamide or p-CMB.
 5. The method according to claim 1,characterized in that said labeling agent is a derivative of saidmodifying agent.
 6. The method according to claim 1, characterized inthat said labeling agent is luminescent and/or contains at least oneluminophor and/or contains a affinity ligand.
 7. The method according toclaim 1, characterized in that said protein has enzymatic activityand/or is a receptor, a transmembrane receptor, a 7-transmembranereceptor, or a soluble protein receptor.
 8. The method according toclaim 1, characterized in that said modifying agent and/or said labelingagent is added in excess.
 9. The method according to claim 1,characterized in that excess modifying agent is removed or inactivatedprior to said incubation with the labeling agent.
 10. The methodaccording to claim 1, characterized in that said specific labeling ofthe protein is detected by a Western blot method.
 11. The methodaccording to claim 1, characterized in that said specific labeling ofthe protein is detected by spectroscopical methods, using a detectionsystem based on confocal fluorescence spectroscopy, fluorescencecorrelation spectroscopy, and/or near-field spectroscopy.
 12. Ascreening method for determining substances which interact with aprotein, wherein said protein and said substance are employed in themethod according to claim 1 and the labeling of the protein is detected.13. A screening method for determining proteins which interact with asubstance, wherein said proteins and said substance are employed in themethod according to claim 1 and the labeling of the protein is detected.14. A method for the detection of the activation or deactivation and/orfor determining conformational changes of proteins using substanceswhich interact with said proteins, wherein said proteins and saidsubstances are employed in the method according to claim 1 and thelabeling of the protein is detected.